EP0377114B1 - Verfahren zur Herstellung eines Pertussis-Toxin-Toxoides - Google Patents

Verfahren zur Herstellung eines Pertussis-Toxin-Toxoides Download PDF

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Publication number
EP0377114B1
EP0377114B1 EP89122006A EP89122006A EP0377114B1 EP 0377114 B1 EP0377114 B1 EP 0377114B1 EP 89122006 A EP89122006 A EP 89122006A EP 89122006 A EP89122006 A EP 89122006A EP 0377114 B1 EP0377114 B1 EP 0377114B1
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EP
European Patent Office
Prior art keywords
pertussis toxin
toxoid
concentration
lysine
treatment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP89122006A
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English (en)
French (fr)
Other versions
EP0377114A1 (de
Inventor
Akihiro Ginnaga
Kazunori Morokuma
Katsutoshi Aihara
Mitsuo Sakoh
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chemo Sero Therapeutic Research Institute Kaketsuken
Original Assignee
Chemo Sero Therapeutic Research Institute Kaketsuken
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/099—Bordetella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/82—Proteins from microorganisms
    • Y10S530/825—Bacteria

Definitions

  • the present invention relates to a method for toxoidizing pertussis toxin, more particularly, to a novel method for preparing pertussis toxin toxoid from pertussis toxin by which the toxoid will not exhibit toxicity reversion, thereby making the toxoid highly suitable for use in pertussis vaccines having no substantial side effects. Furthermore, the invention relates to pertussis toxin toxoids obtainable according to said method and to vaccines containing said pertussis toxin toxoid.
  • Pertussis toxin is a protein produced by Bordetella pertussis and, because of the variety of its physiological and biological activities, is also referred to as leukocytosis promoting factor (LPF), histamine sensitizing factor (HSF), or islets activating protein (IAP).
  • LPF leukocytosis promoting factor
  • HSF histamine sensitizing factor
  • IAP islets activating protein
  • pertussis toxin is known to exhibit such biological activities as promotion of vascular permeability, CHO-cell clustering activity , and ADP-ribosylase activity.
  • pertussis toxin is one of the most important antigens against infection with B. pertussis.
  • the technical problem underlying the present invention therefore is to overcome the above-mentioned problems and to provide a novel method for preparing pertussis toxin toxoid from pertussis toxin by which the resultant toxoid is completely devoid of toxicity and does not exhibit toxicity reversion while retaining the sufficient antigenicity. Furthermore, the technical problem underlying the present invention is to provide new pertussis toxin toxoids and vaccines containing them.
  • the solution of said technical problem is based on the finding that, in the treatment of pertussis toxin with formaldehyde (formalin) for the preparation of the toxoid, the presence of specific amino acids provides sufficient inactivation of the toxin without possibility of the toxicity reversion.
  • a method for preparing pertussis toxin toxoid by a treatment of pertussis toxin with formaldehyde which comprises carrying out said treatment in the presence of lysine or glycine in combination with one or more of amino acids selected from the group consisting of N-acetyl-DL-tryptophan, N-acetyl-D-tryptophan and N-acetyl-L-tryptophan.
  • the present invention provides pertussis toxin toxoids obtainable according to said method and the corresponding vaccines. These vaccines optionally also contain pharmaceutically acceptable carriers, excipients add/or diluents.
  • the present invention is based on the fact that the treatment of pertussis toxin with formaldehyde in the presence of N-acetyltryptophan together with lysine or glycine can produce toxoids which are much superior to those obtained by conventional detoxification methods including the ones described in the above-mentioned earlier application by the present inventors (EP-A 0 121 249) in which there are employed other amino acid(s) than those defined in the present invention. It is particularly notable that the pertussis toxin toxoid prepared by the method of the present invention does not exhibit substantial toxicity reversion even under severe conditions, as evidenced by an assay system recently developed for sensitive analysis.
  • the method of the present invention for the preparation of the toxoid is generally carried out with 0.2 to 1.2% of formalin for 5 to 50»g of pertussis toxin in terms of PN(protein nitrogen)/ml, in which the concentration of N-acetyltryptophan is in the range of 0.1 to 10mM, the concentration of lysine is in the range of 5 to 200mM and the concentration of glycine is in the range of 5 to 200mM, at a temperature of 20 to 45°C for a treatment period of 3 to 30 days. Between lysine and glycine, lysine is preferable.
  • the method of the present invention is practiced preferably with the formalin concentration being 0.3 to 1.0% (most preferably 0.6 to 0.8%), N-acetyltryptophan concentration being 0.2 to 4mM (most preferably 0.5 to 2mM), lysine concentration being 20 to 100mM (most preferably, 10 to 50mM), glycine concentration being 5 to 120mM (most preferably 20 to 40mM), and the temperature being 25 to 45°C (most preferably 37 to 42°C ) for a period of 7 to 21 days (most preferably 7 to 10 days).
  • the various physiological and biological activities possessed by the pertussis toxin are reliably inactivated for detoxification, while the immunogenicity is retained as can be confirmed by the mouse potency test.
  • the pertussis toxin toxoid obtained by the present invention does not demonstrate substantial change in the degrees of the physiological and biological activities and does not produce toxicity reversion, even when maintained at 37°C for a period of several weeks. Accordingly, the method of the present invention for the preparation of the toxoid is of outstanding utility for providing efficacious and safe pertussis vaccines.
  • the toxoids (Sample Nos. 1,2,8 and 9) prepared according to the method of the present invention exhibited no toxic activities while possessing high potencies, as can be seen from the data on the physiological and biological activities given in the table. Particularly notable is that the toxoids obtained by the present invention demonstrated no toxic activities and no toxicity reversion even when maintained at 37°C for four weeks. Such effect is particularly notable when N-acetyltryptophan and lysine are added.
  • samples subjected to a single addition of N-acetyltryptophan, lysine or glycine showed a tendency toward toxicity reversion as such toxoid samples still exhibited some extent of physiological and biological activities when maintained at 37°C.
  • the treatment with formalin alone does not provide satisfactory inactivation of the physiological and biological activities. This is particularly the case when the sample was maintained at 37°C, since the results show that the substantial levels of the physiological and biological activities for toxicity are still maintained.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Claims (6)

  1. Verfahren zur Herstellung eines Pertussistoxin-Toxoids durch Behandlung von Pertussistoxin mit Formaldehyd, umfassend die Durchführung der Behandlung in Gegenwart von Lysin oder Glycin in Kombination mit mindestens einer der Aminosäuren N-Acetyl-DL-tryptophan, N-Acetyl-D-tryptophan oder N-Acetyl-L-tryptophan.
  2. Verfahren nach Anspruch 1, wobei 0,2 bis 1,2 % Formalin pro 5 bis 50 »g Pertussistoxin bezogen auf PN (Proteinstickstoff)/ml verwendet werden, die Konzentration an N-Acetyltryptophan im Bereich von 0,1 bis 10 mM, die Konzentration von Lysin im Bereich von 5 bis 200 mM, die Konzentration von Glycin im Bereich von 5 bis 200 mM liegt, die Temperatur 20 bis 45°C ist und der Behandlungszeitraum 3 bis 30 Tage beträgt.
  3. Verfahren nach Anspruch 2, wobei die Formalinkonzentration 0,3 bis 1,0 %, die N-Acetyltryptophan-Konzentration 0,5 bis 2 mM, die Lysinkonzentration 10 bis 50 mM, die Glycinkonzentration 20 bis 40 mM beträgt, die Temperatur 37 bis 42°C ist und der Behandlungszeitraum 7 bis 10 Tage beträgt.
  4. Verfahren nach einem der Ansprüche 1 bis 3, wobei Lysin verwendet wird.
  5. Pertussistoxin-Toxoid, erhältlich gemäß einem Verfahren nach einem der Ansprüche 1 bis 4.
  6. Impfstoff, enthaltend ein Pertussistoxin-Toxoid nach Anspruch 5 und gegebenenfalls zusätzlich einen pharmazeutisch verträglichen Träger, Excipient und/oder Verdünnungsmittel.
EP89122006A 1988-11-29 1989-11-29 Verfahren zur Herstellung eines Pertussis-Toxin-Toxoides Expired - Lifetime EP0377114B1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP303767/88 1988-11-29
JP63303767A JP2706792B2 (ja) 1988-11-29 1988-11-29 百日咳毒素のトキソイド化法

Publications (2)

Publication Number Publication Date
EP0377114A1 EP0377114A1 (de) 1990-07-11
EP0377114B1 true EP0377114B1 (de) 1994-08-24

Family

ID=17925036

Family Applications (1)

Application Number Title Priority Date Filing Date
EP89122006A Expired - Lifetime EP0377114B1 (de) 1988-11-29 1989-11-29 Verfahren zur Herstellung eines Pertussis-Toxin-Toxoides

Country Status (6)

Country Link
US (1) US4996299A (de)
EP (1) EP0377114B1 (de)
JP (1) JP2706792B2 (de)
AT (1) ATE110279T1 (de)
CA (1) CA2004206C (de)
DE (1) DE68917709T2 (de)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006002502A1 (en) * 2004-07-05 2006-01-12 Fundação Butantan Process for obtention of new cellular pertussis vaccine

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5165927A (en) * 1986-08-01 1992-11-24 University Of Southern California Composition with modified pertussis toxin
US5332583A (en) * 1987-11-24 1994-07-26 Connaught Laboratories Limited Vaccine containing genetically-detoxified pertussis holotoxin
US5578308A (en) * 1990-02-12 1996-11-26 Capiau; Carine Glutaraldehyde and formalin detoxified bordetella toxin vaccine
GB9021004D0 (en) * 1990-09-27 1990-11-07 Wellcome Found Acellular vaccines
GB9216351D0 (en) * 1992-07-31 1992-09-16 Wellcome Found Vaccine production
JP5207380B2 (ja) * 2006-03-27 2013-06-12 北里第一三共ワクチン株式会社 長期保存においても毒性復帰がおこらない特徴を持つ全菌体細菌ワクチンならびにその用途

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5750925A (en) * 1980-09-12 1982-03-25 Takeda Chem Ind Ltd Preparation of pertussis toxoid
CA1213234A (en) * 1983-03-30 1986-10-28 Akihiro Ginnaga Method for the production of ha fraction containing protective antigens of bordetella pertussis and pertussis vaccine
KR890001003B1 (ko) * 1984-07-19 1989-04-18 자이단 호오진 까가꾸 오요비 겟세이 료오호 겐뀨쇼 Lpf-ha의 정제방법
GB8512972D0 (en) * 1985-05-22 1985-06-26 Univ Glasgow Vaccine production
GB8601279D0 (en) * 1986-01-20 1986-02-26 Public Health Lab Service Purification of pertussis antigens
FR2597344B1 (fr) * 1986-04-16 1989-06-23 Merieux Inst Perfectionnement au procede de purification d'antigenes proteiques de bacteries appartenant au genre bordetella, en vue de l'obtention d'un vaccin acellulaire.
US4762710A (en) * 1986-06-16 1988-08-09 The United States Of America As Represented By The Department Of Health And Human Services Novel method of preparing toxoid by oxidation and metal ions
US4845036A (en) * 1987-02-03 1989-07-04 The United States Of America As Represented By The Department Of Health And Human Services Process for isolation of the B oligomer of pertussis toxin
CA1337859C (en) * 1987-04-24 1996-01-02 Masashi Chazono Method for culturing bordetella pertussis, a pertussis toxoid and a pertussis vaccine

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006002502A1 (en) * 2004-07-05 2006-01-12 Fundação Butantan Process for obtention of new cellular pertussis vaccine

Also Published As

Publication number Publication date
CA2004206A1 (en) 1990-05-29
CA2004206C (en) 2000-02-08
US4996299A (en) 1991-02-26
DE68917709D1 (de) 1994-09-29
ATE110279T1 (de) 1994-09-15
DE68917709T2 (de) 1995-05-04
JP2706792B2 (ja) 1998-01-28
JPH02149529A (ja) 1990-06-08
EP0377114A1 (de) 1990-07-11

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